The MTT assay is widely used in cell proliferation and cytotoxicity assays. Most cellular bioreduction of MTT is associated with enzymes of the endoplasmic reticulum and involves the reduced pyridine nucleotides NADH and, to a lesser extent, NADPH. Succinate is a weak electron donor for mitochondrial MTT reduction. Recently, new tetrazolium salt assays that use intermediate electron acceptors to facilitate reduction have been introduced. In this article, we compare the biochemical basis of reduction of MTT, XTT, and WST-1. Unlike MTT, XTT and WST-1 are efficiently reduced by NADH and NADPH in the absence of cells or enzymes, and their reduction involves superoxide. Cellular reduction of WST-1 occurs at the cell surface and also involves superoxide.

The Biochemical and Cellular Basis of Cell Proliferation Assays That Use Tetrazolium Salts